Manipulation of microfluidic droplets

ABSTRACT

The invention provides methods for assessing one or more predetermined characteristics or properties of a microfluidic droplet within a microfluidic channel, and regulating one or more fluid flow rates within that channel to selectively alter the predetermined microdroplet characteristic or property using a feedback control.

CROSS-REFERENCE To RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/162,521, filed Mar. 23, 2009, the contents of which are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to the control and manipulation of microdropletswithin microchannels.

BACKGROUND OF THE INVENTION

Methods for generating microdroplets of a uniform volume at a regularfrequency are well known in the art. However, sample to samplevariations in viscosity, viscoelasticity, surface tension or otherphysical properties of the sample fluid coming from, but not limited to,the inclusion of polymers, detergents, proteins, cells, nucleic acids orbuffering solutions, influence the droplet size and volume and, hence,the frequency of generation in an unpredictable way. Thus, the samenozzle on the same microfluidic substrate with same carrier fluid, but adifferent dispersed fluid will result in a different droplet volume at adifferent frequency. These limitations also have an impact on the extentto Which volumes can be reproducibly combined. Together with typicalvariations in pump flow rate precision and variations in channeldimensions, microfluidic systems are severely limited without a means tocompensate on a run-to-run basis.

As a result of the above factors, current microdroplet technologiescannot efficiently or reliably be used for applications involvingcombining droplets of different species at high frequencies.Consequently, there is a need in the art for methods of precise control,manipulation and regulation of droplet frequency generation, frequencyof library droplet introduction and droplet volume.

SUMMARY OF THE INVENTION

The present invention provides a feedback control system formicrofluidic droplet manipulation comprising: providing a microfluidicsystem comprising at least one microfluidic channel containing at leastone fluidic droplet; detecting at least one predeterminedcharacteristics of said fluidic droplet at one or more positions withinsaid microfluidic channel; assessing said predetermined characteristicusing an image sensor; and transmitting said assessment from said imagesensor to a feedback controller, wherein said feedback controlleradjusts a flow rate of one or more fluids, thereby manipulating saidfluidic droplet within said microfluidic channel. The detecting at leastone predetermined characteristics of said fluidic droplet at one or morepositions within said microfluidic channel can further comprisesacquiring a plurality of images of said fluidic droplet at a pluralityof time points within said microfluidic channel, wherein said pluralityof images comprises an image set. The system can further include:assessing said predetermined characteristic of said fluidic droplet insaid microfluidic channel, within each image set, using an image sensor;comparing said assessment of said predetermined characteristic of saidfluidic droplet in each image set; and determining an average assessmentof said predetermined characteristic of said fluidic droplet; whereinsaid feedback controller adjusts a flow rate of one or more fluids,thereby increasing the accuracy of the assessment.

The predetermined characteristic can be droplet volume, dropletgeneration rate, droplet arrival frequency, droplet release rate, ortotal droplet count. The one or more fluids can be a carrier fluid or adrive fluid,

The present invention also provides a feedback control system formanipulating microfluidic droplet pairing ratios comprising: providing amicrofluidic system comprising at least one microfluidic channel;producing a first plurality of fluidic droplets within said microfluidicchannel at a first frequency; producing a second plurality of fluidicdroplets within said microfluidic channel at a second frequency, whereinat least one fluidic droplet from said first plurality and at least onefluidic droplet from said second plurality are paired; assessing saidfirst frequency and said second frequency using an image sensor; andtransmitting said assessment of said first and said second frequencyfrom the image sensor to a feedback controller; wherein said feedbackcontroller adjusts a flow rate of one or more fluids to provide adesired frequency ratio of said first to said second plurality ofdroplets, thereby manipulating the pairing ratios of said first andsecond pluralities of fluidic droplets within said microfluidic channel.The first plurality of fluidic droplets and the second plurality offluidic droplets were introduced at the same frequency and wherein saidfeedback controller adjusts a flow rate of one or more fluids tomaintain said first and said second frequency at the same frequency.

The first and second pluralities of fluidic droplets can differ in size,color, refractive index, or extinction coefficient. The first and secondpluralities of fluidic droplets can contain a different biological,biochemical, or chemical entity. The desired frequency ratio of thefirst plurality of droplets to the second plurality of droplets can be1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. Preferably, thedesired frequency ratio of the first plurality of droplets to the secondplurality of droplets is 1:1.

The present invention also provides a feedback control system forcontrolling microfluidic droplet count comprising: providing amicrofluidic system comprising at least one microfluidic channel;producing at least a first plurality of fluidic droplets within saidmicrofluidic channel at a first frequency; assessing said firstfrequency using an image sensor; determining the time required toproduce a predetermined amount of fluidic droplets based upon saidfrequency assessment; and transmitting said assessment to a feedbackcontroller, wherein said feedback controller stops said introduction ofsaid droplets after said determined time, thereby controlling themicrofluidic droplet count.

The present invention also provides a feedback control system forindependently controlling microfluidic droplet volume and frequencycomprising: providing a microfluidic system comprising at least onemicrofluidic channel; producing a plurality of fluidic droplets within acarrier fluid within said microfluidic channel using a drive fluid;assessing the frequency, volume, and flow rate of said plurality ofdroplets using an image sensor; transmitting said assessed frequencies,volumes, and flow rates of the plurality of droplets front said imagesensor to a feedback controller; adjusting a flow rate of the carrierfluid using said feedback controller to attain a predetermined dropletfrequency set point; and adjusting a flow rate of the drive fluid usingsaid feedback controller to attain a predetermined droplet volume setpoint; wherein said feedback control system independently determines andcontrols microfluidic droplet frequency and volume. The plurality offluidic droplets can be generated within the microfluidic channel. Theplurality of fluidic droplets can be pre-formed and introduced to themicrofluidic channel.

The invention provides a feedback control system for microfluidicdroplet manipulation including: (a) detecting one or more predeterminedcharacteristics of a droplet at one or more positions within amicrofluidic channel; (b) assessing the predetermined characteristicusing an image sensor; and (c) transmitting the assessment from theimage sensor to a feedback controller, wherein the feedback controlleradjusts a flow rate of one or more fluids, thereby manipulating thedroplet within the microfluidic channel. In one aspect of this system,the predetermined characteristic is droplet volume, droplet generationrate, droplet release rate, or total droplet count. Preferably, thepredetermined characteristic is droplet volume. In another aspect ofthis system, the fluid is a carrier fluid or a drive fluid.

The invention also provides a feedback control system for manipulatingmicrofluidic droplet pairing ratios including: (a) producing a firstplurality of droplets within a microfluidic channel at a frequency; (b)assessing the frequency of the first-plurality of droplets using animage sensor; (c) producing a second plurality of droplets within amicrofluidic channel at the same frequency as the first plurality ofdroplets; (d) assessing the frequency of the second plurality ofdroplets using an image sensor; and (e) transmitting the frequencies ofthe first and second pluralities of droplets from the image sensor to afeedback controller; wherein the feedback controller adjusts a flow rateof one or more fluids to maintain the first and second pluralities ofdroplets at identical frequencies, thereby manipulating the pairingratios of the first and second pluralities of droplets within themicrofluidic channel. In one aspect of this system, the first and secondpluralities of droplets differ in size, color, refractive index, orextinction coefficient. Alternatively, or in addition, the first andsecond pluralities of droplets contain a different biological,biochemical, or chemical entity. In another aspect of this system, thefluid is a carrier fluid or a drive fluid.

Furthermore, the invention provides a feedback control system forassessing and manipulating a predetermined characteristic of amicrofluidic droplet including: (a) acquiring a plurality of images of adroplet at a plurality of time points within a microfluidic channel,wherein said plurality of images comprises an image set; (b) assessingthe predetermined characteristic of the droplet in the microfluidicchannel using an image sensor; and (c) transmitting the assessment fromthe image sensor to a feedback controller, wherein the feedbackcontroller adjusts a flow rate of one or more fluids, therebymanipulating the predetermined characteristic of the droplet within themicrofluidic channel. In one aspect, this system further includes: (a)acquiring a plurality of image sets at a plurality of time points; (b)assessing the predetermined characteristic of the droplet in themicrofluidic channel, within each image set, using an image sensor; (c)comparing the assessment of the predetermined characteristic of thedroplet in each image set; and (d) determining an average assessment ofthe predetermined characteristic of the droplet; wherein the feedbackcontroller adjusts a flow rate of one or more fluids, thereby increasingthe accuracy of the assessment. In another aspect of this system, thepredetermined characteristic is droplet arrival frequency or dropletvolume. Moreover, the fluid of this system is a carrier fluid or a drivefluid.

The invention provides a feedback control system for independentlycontrolling microfluidic droplet volume and frequency including: (a)producing a plurality of droplets within a microfluidic channel; (b)assessing the droplet frequency, volume, and flow rate of the pluralityof droplets using an image sensor; (c) transmitting the frequencies,volumes, and flow rates of the plurality of droplets from the imagesensor to a feedback controller; (d) adjusting a flow rate of thecarrier fluid using a feedback controller to attain a predetermineddroplet frequency set point; and (e) adjusting a flow rate of a drivefluid using a feedback controller to attain a predetermined dropletvolume set point; wherein the feedback control system independentlydetermines microfluidic droplet frequency and volume.

The invention further provides a feedback control system formanipulating microfluidic droplet pairing ratios including: (a)producing a first plurality of droplets within a microfluidic channel ata frequency; (b) assessing the frequency of the first-plurality ofdroplets using an image sensor; (c) producing a second plurality ofdroplets within a microfluidic channel at a second frequency; (d)assessing the frequency of the second plurality of droplets using animage sensor; and (e) transmitting the frequencies of the first andsecond pluralities of droplets from the image sensor to a feedbackcontroller; wherein the feedback controller adjusts a flow rate of oneor more fluids to produce a desired frequency ratio of the first to thesecond plurality of droplets, thereby manipulating the pairing ratios ofthe first and second pluralities of droplets within the microfluidicchannel. In one aspect of this system, the desired frequency ratio ofthe first plurality of droplets to the second plurality of droplets is1:1. Alternatively, the desired frequency ratio of the first pluralityof droplets to the second plurality of droplets is selected from thegroup consisting of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and1:10. Alternatively, or in addition, each droplet of the first pluralityof droplets comprises a single element of a genomic library and eachdroplet the second plurality of droplets comprises a single primer pair.

The invention provides a feedback control system for controllingmicrofluidic droplet count including: (a) producing at least a firstplurality of droplets within a microfluidic channel at a frequency; (b)assessing the frequency of the first-plurality of droplets using animage sensor; (c) determining the time required to produce apredetermined amount of droplets based upon the frequency assessment;and (d) transmitting the assessment to a feedback controller, whereinthe feedback controller stops production of the droplets after thedetermined time, thereby controlling the microfluidic droplet count.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the claimed invention. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods and examples areillustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of a series of images of two fluidic dropletspaired within a microfluidic channel. One droplet is from a firstplurality of droplets at one size and the second droplet is from asecond plurality of droplets and a different size than the droplet fromthe first plurality. This representative image shows the droplet fromthe first plurality and the droplet from the second plurality paired ina 1:1 ratio. The top panel shows droplets in the region of interest(ROI) and the bottom panel shows the corresponding “contour accumulatorimage,” in which the grey level intensity corresponds to the number oftimes each pixel was added to the accumulator image. In the bottompanel, all other pixels were assigned the value “0”, which is shown asblack.

FIG. 2 is a photograph of an image of microdroplets shown incross-section, in which the droplets of bigger cross-sectional area werepseudo-colored yellow (light shaded droplets) and the droplets ofsmaller cross-sectional area were pseudo-colored blue (dark shadeddroplets).

FIG. 3 is a schematic diagram of “feedback control,” in which systeminputs are adjusted according to measured system outputs.

FIG. 4 is a graph of the Library to Template Ratio (LTR), also referredto as the droplet pairing ratio, versus time, showing that the dropletpairing ratio is well controlled over a range from 0.4 to 1.75 byadjusting the carrier fluid flow rates.

FIG. 5A is a pair of graphs, a line graph and its correspondinghistogram, of the relationship of the LTR for open loop operation (witha rather large CV of 8.5%) versus time, showing that the output, or LTR,is not centered about the set point of 1.

FIG. 5B is a pair of graphs, a line graph and its correspondinghistogram, of the relationship of LTR for closed loop feedback (with aCV of 3.0%) versus time, showing that the output, or LTR, is centeredabout the set point of 1.

FIG. 6 is a pair of graphs of droplet diameter (top) or droplet volume(bottom) versus time, demonstrating the droplet volume control stepresponse. Green or Straight Line=set point. Blue or Jagged Line=measuredoutput.

FIG. 7 is a pair of graphs of droplet diameter (top) or droplet volume(bottom) versus time, demonstrating that error in the droplet volume isminimal, i.e. below 0.5 pL.

FIG. 8 is a graph of the measured droplet count vs. droplet count setpoint, demonstrating droplet count control.

FIG. 9A a line-cut along the droplet train of a microfluidic channel.

FIG. 98 is a one-dimensional Fourier transform of a line-cut along thedroplet train (line cut in FIG. 9A) revealing the fundamental dropletfrequency (spatial frequency) and its higher order harmonics.

FIG. 9C is the amplitude of the fundamental frequency after eachincrease in the change in time, ΔT in multiple illumination images.

FIG. 9D is a second Fourier transform of the dependence of thefundamental frequency on ΔT revealing the desired temporal frequency ofthe droplets as a single pronounced peak.

FIG. 10 is an illustration of a trans-illumination scheme.

FIG. 11 is a graph showing that the imaging optics “squeezes” a 2D imageinto a 1D image by optically summing what would be the columns of theblue region of interest into a single line.

FIG. 12 is a graph showing that the average droplet spacing isapproximately 50 pixels and droplet diameter is approximately 25 pixels.

DETAILED DESCRIPTION OF THE INVENTION

The methods of the present invention provide precise and highlyregulated control of microfluidic droplet movement and interactionwithin a microfluidic channel. The invention provides a feedback controlsystem for microfluidic droplet manipulation including: (a) providing amicrofluidic system comprising at least one microfluidic channelcontaining at least one fluidic droplet; (b) detecting at least onepredetermined characteristics of the fluidic droplet at one or morepositions within the microfluidic channel; (c) assessing thepredetermined characteristic using an image sensor; and (d) transmittingthe assessment from the image sensor to a feedback controller, whereinthe feedback controller adjusts a flow rate of one or more fluids,thereby manipulating the fluidic droplet within the microfluidicchannel. The manipulating or controlling a droplet or a plurality ofdroplets within a microfluidic channel includes, but is not limited to,manipulating an absolute or relative droplet volume, a droplet pairingratio, a droplet frequency, a droplet frequency ratio, the number ofdroplets generated and/or a droplet count. The terms manipulating andcontrolling are used interchangeably herein.

The present invention also provides feedback control system forassessing and manipulating a predetermined characteristic of amicrofluidic droplet including: (a) providing a microfluidic systemcomprising at least one microfluidic channel containing at least onefluidic droplet; (b) acquiring a plurality of images of the fluidicdroplet at a plurality of time points within the microfluidic channel,wherein the plurality of images comprises an image set; (c) assessingthe predetermined characteristic of the fluidic droplet in themicrofluidic channel using an image sensor; and (d) transmitting theassessment from the image sensor to a feedback controller, wherein thefeedback controller adjusts a flow rate of one or more fluids, therebymanipulating the predetermined characteristic of the fluidic dropletwithin the microfluidic channel. The system can further include:assessing said predetermined characteristic of said fluidic droplet insaid microfluidic channel, within each image set, using an image sensor;comparing said assessment of said predetermined characteristic of saidfluidic droplet in each image set; and determining an average assessmentof said predetermined characteristic of said fluidic droplet; whereinsaid feedback controller adjusts a flow rate of one or more fluids,thereby increasing the accuracy of the assessment.

Microdroplets are essentially miniaturized test tubes with a volume ofless than 1 pico-liter (one trillionth of a liter) to several hundrednanoliters (one billionth of a liter). Because of their incredibly smallsize, each microdroplet requires only a very small amount of sample toconduct chemical reactions, biological assays and medical testing, thusyielding a wealth of information for biomedical and chemical studiesfrom very limited source material at relatively low cost, e.g., a 10micro-liter sample can be used for 1 million reactions with eachreaction using 10 pico-liters. Furthermore, microdroplets can beintroduced into microfluidic devices, which feature a series ofmicrometer-sized channels etched or molded into a chip wheremicrodroplets can be manipulated by directing the flow of the fluidsthat carry them. The term “carrier fluid” or “carrier fluids” refers toany fluid which contains droplets and transports them throughmicrofluidic channels of microfluidic devices. Carrier fluids aredescribed in greater detail herein.

In microfluidic devices, microdroplets can be processed, analyzed andsorted at a highly efficient rate of several thousand droplets persecond, providing a powerful platform which allows rapid screening ofmillions of distinct compounds, biological probes, proteins or cellseither in cellular models of biological mechanisms of disease, or inbiochemical, or pharmacological assays. Although major improvements inregulating droplet size and uniformity, and modifying droplet surfacechemistry have been achieved, the utility of microdroplets in chemistry,biology, and medicine depends critically on the spatiotemporally precisedelivery of microdroplets of various properties through the channels inmicrofluidic devices.

In order to utilize microdroplets for rapid large-scale chemicalscreening or complex biological library identification, differentspecies of microdroplets, each containing the specific chemicalcompounds or biological probes of interest, have to be generated andcombined at the preferred conditions, e.g., mixing ratio and order ofcombination. For example, one microdroplet of species A must be combinedwith one microdroplet of species B, but not with two microdroplets ofspecies B or with one microdroplet of species C. The ratio of combiningdifferent species of microdroplets is achieved by adjusting thefrequencies at which microdroplets are delivered to the site ofcombination. The terms “frequency” or “frequencies” refer to the rate atwhich microdroplets of certain species are delivered to a specificlocation. Moreover, this frequency or rate is a number per unit time,typically several hundred to tens of thousands per second. Furthermorethe terms “frequency” or “frequencies” refers to the number of times atwhich droplets of certain species are delivered to a specific location.The location can be where certain behaviors of droplets (e.g., pairing,merging, combination, etc.) occur or where certain actions (e.g.,electrification, mechanical deformation, etc.) are applied to droplets.Preferably, the location is where combination of droplets occurs.

Preferably, each species of droplet is introduced at a confluence pointin a main microfluidic channel from separate inlet microfluidicchannels. Preferably, droplet volumes are chosen by design such that onespecies is larger than others and moves at a different speed, usuallyslower than the other species, in the carrier fluid, as disclosed inU.S. Publication No. US 2007/0195127 and International Publication No.WO 2007/089541, each of which are incorporated herein by reference intheir entirety. The channel width and length is selected such thatfaster species of droplets catch up to the slowest species. Sizeconstraints of the channel prevent the faster moving droplets frompassing the slower moving droplets resulting in a train of dropletsentering a merge zone. In the merge zone, droplets are induced tocoalesce into a single droplet, preferably an electric field is utilizedto induce coalescence. Multi-step chemical reactions, biochemicalreactions, or assay detection chemistries often require a fixed reactiontime before species of different type are added to a reaction.Multi-step reactions are achieved by repeating the process multipletimes with a second, third or more confluence points each with aseparate merge point. Highly efficient and precise reactions andanalysis of reactions are achieved when the frequencies of droplets fromthe inlet channels are matched to an optimized ratio and the volumes ofthe species are matched to provide optimized reaction conditions in thecombined droplets.

Key elements for using microfluidic channels to process dropletsinclude: (1) producing droplet of the correct volume, (2) producingdroplets at the correct frequency and (3) bringing together a firststream of sample droplets with a second stream of sample droplets insuch a way that the frequency of the first stream of sample dropletsmatches the frequency of the second stream of sample droplets.Preferably, bringing together a stream of sample droplets with a streamof premade library droplets in such a way that the frequency of thelibrary droplets matches the frequency of the sample droplets.

Methods for producing droplets of a uniform volume at a regularfrequency are well known in the art. One method is to generate dropletsusing hydrodynamic focusing of a dispersed phase fluid and immisciblecarrier fluid, such as disclosed in U.S. Publication No. US 2005/0172476and International Publication No. WO 2004/002627. Feedback on theinfusion rates of the carrier fluid and the dispersed fluid providesdroplets that are uniform in size and generated at a fixed frequencyover arbitrarily long periods of time. However, sample to samplevariations in viscosity, viscoelasticity, surface tension or otherphysical properties of the sample fluid coming from but not limited tothe inclusion of polymers, detergents, proteins, cells, nucleic acids orbuffering solutions, influence the droplet size, and, hence, frequencyof generation in an unpredictable way, generating a significant problemto be solved. Hence, the same nozzle on the same substrate with samecarrier fluid, but a different dispersed fluid will result in adifferent droplet volume at a different frequency. Moreover, often it isdesirable for one of the species introduced at the confluence to be apre-made library of droplets where the library contains a plurality ofreaction conditions, e.g., a library can contain plurality of differentcompounds at a range of concentrations encapsulated as separate libraryelements for screening their effect on cells or enzymes, alternatively alibrary could be composed of a plurality of different primer pairsencapsulated as different library elements for targeted amplification ofa collection of loci, alternatively a library could contain a pluralityof different antibody species encapsulated as different library elementsto perform a plurality of binding assays. The introduction of a libraryof reaction conditions onto a substrate is achieved by pushing a premadecollection of library droplets out of a vial with a drive fluid. Thedrive fluid is a continuous fluid. The drive fluid may comprise the samesubstance as the carrier fluid (e.g., a fluorocarbon oil). For example,if a library consists of ten pica-liter droplets is driven into a inletchannel on a microfluidic substrate with a drive fluid at a rate of10,000 pico-liters per second, then nominally the frequency at which thedroplets are expected to enter the confluence point is 1000 per second.However, in practice droplets pack with oil between them that slowlydrains. Over time the carrier fluid drains from the library droplets andthe number density of the droplets (number/mL) increases. Hence, asimple fixed rate of infusion for the drive fluid does not provide auniform rate of introduction of the droplets into the microfluidicchannel in the substrate. Moreover, library-to-library variations in themean library droplet volume result in a shift in the frequency ofdroplet introduction at the confluence point. Thus, the lack ofuniformity of droplets that results from sample variation and oildrainage provides another problem to be solved. For example if thenominal droplet volume is expected to be 10 pica-liters in the library,but varies from 9 to 11 pico-liters from library-to-library then a10,000 pico-liter/second infusion rate will nominally produce a range infrequencies from 900 to 1,100 droplet per second. In short, sample tosample variation in the composition of dispersed phase for droplets madeon chip, a tendency for the number density of library droplets toincrease over time and library-to-library variations in mean dropletvolume severely limit the extent to which frequencies of droplets can bereliably matched at a confluence by simply using fixed infusion rates.In addition, these limitations also have an impact on the extent towhich volumes can be reproducibly combined. Combined with typicalvariations in pump flow rate precision and variations in channeldimensions, systems are severely limited without a means to compensateon a run-to-run basis. The foregoing facts not only illustrate problemto be solved, but also demonstrate a need for a method of instantaneousregulation of microfluidic control over microdroplets within amicrofluidic channel.

As a result of the above factors, current microdroplet technologiescannot efficiently or reliably be used for applications involvingcombining droplets of different species at high frequencies.Consequently, there is a need in the art for novel methods ofmanipulating droplet frequency of generation, frequency of librarydroplet introduction and droplet volume.

It is well established to one of ordinary skill in the art that objectsand geometrical properties of objects are identified from standard imageacquisition and machine vision protocols. For example, objects in imagesof microfluidic channels such as droplets, channel walls, orcontaminating particulate are readily distinguished and classified bytheir boundary, projected area, and ellipticity of the objects.

The invention provides a method for capturing images of objects withinmicrofluidic channels such as microdroplets and channel walls,collecting the information to measure and assess both frequency andvolume, and subsequently changing the infusion rates to match specificset points. The benefit of using image processing to measure dropletparameters in-situ allows system requirements such as pump flow rateaccuracy and microfluidic channel tolerances to be relaxed. Thus, imageprocessing protocols provide the practical advantage of reducing thesystem cost.

The invention provides a feedback control system for microfluidicdroplet manipulation of one or more predetermined properties orcharacteristics of a microdroplet. One embodiment of the invention isdirected to a system for dynamically measuring or assessing, andcontrolling or manipulating droplets via machine vision for feedbackmeasurement and adjusting fluid flow rates to manipulate one or morepredetermined properties or characteristics of a microdroplet. Examplesof controllable droplet properties or characteristics include, but arenot limited to, droplet volume, droplet generation rate, droplet releaserate, and the total number of droplets generated. Preferably theselective manipulation occurs with droplets in a microfluidic device.Such microfluidic devices are generally known in the art. Exemplarypreferred microfluidic devices are provided by U.S. Publication No. US2008/0003142, international Publication No. WO 2008/063227, U.S.Publication No. US 2008/0014589, and International Publication No. WO2007/081385, each of which are incorporated herein by reference in theirentirety. Flow rates are adjusted by a drive infusion system that is notconstrained to a defined technology or mechanism. Methods of theinvention encompass art-recognized drive infusion systems, includingthose systems disclosed in U.S. Publication No. US 2008/0003142,International Publication No. WO 2008/063227, U.S. Publication No. US2008/0014589, and International Publication No. WO 2007/081385.Furthermore, exemplary drive infusion systems of the methods of theinvention include, but are not limited to, a syringe pump, pressurehead, electrokinetic drive or any other means known in the art.

“Feedback control,” as shown in FIG. 3, refers to adjusting systeminputs according to measured, assessed, characterized, or determinedsystem outputs. Exemplary system outputs include, but are not limitedto, the image processing LTR measurement, an assessment from an imagescanner (a measurement of size, speed, frequency, refractive index,extinction coefficient, color, volume, area, number, phase, coalescence,or a determination of the contents of a micro-fluidic droplet), acharacteristic or property of a microfluidic droplet or plurality ofdroplets (size, speed, frequency, refractive index, extinctioncoefficient, color, volume, area, number, phase, coalescence, content oractivity thereof, fluorescence, or any change thereof), a characteristicor property of a fluid within a microfluidic channel (content,viscosity, surface tension, clarity, opacity, thickness, shear forces,speed, volume, pressure, temperature, and solubility), and acharacteristic or property of the microfluidic device itself. Exemplarysystem inputs include, but are not limited to, a microfluidic droplet ora plurality of microfluidic droplets, one or more fluids, automatedinstructions transmitted to one or more pumps or devices that control afluid within a microfluidic device, or automated instructionstransmitted to one or more pumps or devices that control to introductionof droplets into a microfluidic channel or the production, generation,or creation of a microfluidic droplet within a microfluidic channel of amicrofluidic device. System outputs are assessed, and signals orinstructions are transmitted from a feedback controller to a device thatcontrols a system input. The feedback controller adjusts system inputeither in response to changing system outputs to maintain a constantstate of efficiency or to manipulate a microfluidic droplet or pluralityof droplets.

The present invention provides methods to selectively measure or assessand manipulate the absolute or relative droplet volume. The relativedroplet volume can be determined by analysis of an image captured by animage scanner. This analysis includes capturing an image of a droplet,or a plurality of droplets, at a point in a microfluidic channelcontaining a lithographically inscribed size marker, such as a circle ora square; determining the number of image pixels occupied by a dropletand by the size marker; and comparing the resultant pixel numbers todetermine a relative droplet volume. Absolute droplet volume isdetermined by dividing a flow rate, such as the infusion flow rate,represented as

by the droplet frequency, represented by v, in the following equation:

$\overset{\_}{V} = {\frac{Q}{v}.}$

In one example, the droplet volume is controlled by adjusting the drivefluid through feedback control based on the droplet projected area asmeasured by an image sensor. In a preferred embodiment of this method,the image sensor is a digital image sensor. In another example, thedroplet volume is controlled by adjusting the drive fluid throughfeedback control based upon the droplet volume, as measured or assessedby Pulsed Illumination Scanning (PILS).

The present invention also provides methods to selectively manipulatedroplet pairing ratios. The present invention provides a feedbackcontrol system for manipulating microfluidic droplet pairing ratiosincluding: (a) providing a microfluidic system comprising at least onemicrofluidic channel; (b) introducing a first plurality of fluidicdroplets within the microfluidic channel at a first frequency; (c)introducing a second plurality of fluidic droplets within themicrofluidic channel at a second frequency, wherein at least one fluidicdroplet from the first plurality and at least one fluidic droplet fromthe second plurality are paired; (d) assessing the first frequency andthe second frequency using an image sensor; and (e) transmitting theassessment of the first and the second frequency from the image sensorto a feedback controller; wherein the feedback controller adjusts a flowrate of one or more fluids to maintain the first and the secondfrequency at the same frequency, thereby manipulating the pairing ratiosof the first and second pluralities of fluidic droplets within themicrofluidic channel. The present invention also provides a feedbackcontrol system for manipulating microfluidic droplet pairing ratiosincluding: (a) providing a microfluidic system comprising at least onemicrofluidic channel; (b) introducing a first plurality of fluidicdroplets within the microfluidic channel at a first frequency; (c)introducing a second plurality of fluidic droplets within themicrofluidic channel at a second frequency; (d) assessing the firstfrequency and the second frequency using an image sensor; and (e)transmitting the assessment of the first and the second frequency fromthe image sensor to a feedback controller; wherein the feedbackcontroller adjusts a flow rate of one or more fluids to provide adesired frequency ratio of the first to the second plurality ofdroplets, thereby manipulating the pairing ratios of the first andsecond pluralities of fluidic droplets within the microfluidic channel.

The frequencies of a first droplet and a second droplet, or a firstplurality and a second plurality of droplets, are controlled relative toeach other to have the same frequency but out of phase such that thedroplets are intercalated, or interdigitated, (and thus paired) whentraveling through the microfluidic channel. A first plurality ofdroplets and a second plurality of droplets having identical or matchedfrequencies, and which enter a microfluidic channel at the same time,are out-of-phase when either the first or second plurality of dropletstravel down the microfluidic channel at a different speed from theother. As such, the droplets of the first and second pluralitiesintercalate, or interdigitate, because they do not travel together. In apreferred embodiment, the frequencies of the first and secondpluralities are not identical, but rather matched, such, thatintercalation, or interdigitation, of the droplets still occurs. Forexample, the frequency of a second plurality of droplets that is matchedto the frequency of a first plurality of droplets is greater to or lessthan the frequency of the first plurality by approximately 1, 10, 100,or 1000 Hz, or any point in between.

The present invention further provides methods to selectively manipulatethe number of droplets generated. In one example, the system counts thenumber of droplets generated and stops pump flow once the desired numberof droplets is reached. Thus, the present invention provides a feedbackcontrol system for controlling microfluidic droplet count including: (a)providing a microfluidic system comprising at least one microfluidicchannel; (b) introducing at least a first plurality of fluidic dropletswithin the microfluidic channel at a first frequency; (c) assessing thefirst frequency using an image sensor; (d) determining the time requiredto produce a predetermined amount of fluidic droplets based upon thefrequency assessment; and (e) transmitting the assessment to a feedbackcontroller, wherein the feedback controller stops the introduction ofthe droplets after the determined time, thereby controlling themicrofluidic droplet count.

The present invention provides a process including droplet detection,droplet assessment and characterization, and feedback control, forselectively manipulating the various droplet properties orcharacteristics in a microfluidic device.

Machine vision provides a means to accurately detect and characterizeproperties of droplets. Droplet characterization is then used to adjustthe fluidic system inputs, fluid flow rates and drive infusion flowrates to manipulate the droplet characteristics or properties. Thesecharacterization and control schemes are applied in parallel, forexample frequency, droplet diameter and droplet pairing are controlledat the same time. Alternatively, these characterization and controlschemes are applied in series, for example frequency, droplet diameterand droplet pairing are controlled sequentially.

The invention provides a method for measuring and controlling thearrival frequency of regularly separated objects, e.g. droplets,including the measurement of multiple images acquired at different times(e.g. image sets) to measure the displacement of the objects, andacquisition of different image sets at varying times between images toreduce the uncertainty in the measurement. Methods of the inventionaccurately and inexpensively measure droplet frequency and volume. Thepresent invention provides methods to selectively manipulate thefrequency of droplets generated and released by adjusting the flow rateof a fluid, for example, the carrier fluid or drive fluid. In oneexample, the flow rate of the carrier fluid and drive fluid is adjustedin response to detecting the distance a single droplet moves during aknown quantity of time, e.g. as determined by Pulsed IlluminationScanning.

“Droplet pairing” refers to the process of interleaving differentclasses of droplets at a time variant ratio (e.g. user settable functionor constant value). The ratio is defined as x droplets of species A forevery droplet of species B. In one example, two different classes ofdroplets are intercalated, or interdigitated, wherein the dropletsdiffer in size (e.g., diameter, perimeter, diagonal, volume, area ofcross-section etc), shape (e.g., spherical, elliptical, rectangular,etc.), color, refractive index or extinction coefficient. The term“refractive index” refers to the ability of a medium (e.g., glass, air,solution, etc.) to reduce the speed of waves (e.g., light, radio wave,sound wave, etc.) traveling inside the medium. The term “extinctioncoefficient” refers to the strength of a medium (e.g., glass, air,solution, etc.) to absorb or scatter light. The term “cross-section”refers to the intersection of a body in 2-dimensional space with a line,or of a body in 3-dimensional space with a plane. Preferably,cross-section refers to the intersection of a body in 3-dimensionalspace with a plane.

In a further example, the two classes of droplets have differentdiameters. All droplets in the microfluidic device within the ROI aredetected using the previously specified droplet detection algorithm. Thedroplets are further classified as species A or species B depending onthe droplet area. The droplet pairing ratio is measured by counting thenumber of species A droplets that are found upstream of each species Bdroplet. Species A has a smaller droplet diameter and travels fasterthan species B. Only the upstream Species A droplets will merge withdownstream Species B droplets due to the differences in velocity. Thespecies A droplets corresponding to a species B droplet at the inlet ofthe microfluidic channel are not counted in the droplet pairingmeasurement as it is not possible to detect and classify the offimage-frame upstream droplet to get an exact pairing ratio for thatspecies A:species B droplet set.

As shown in FIG. 4, the droplet pairing ratio (also referred to asLibrary to Template Ratio [LTR]) is well controlled over a range from0.4 to 1.75 by adjusting the carrier fluid flow rates. FIG. 5a shows theLTR for open loop operation with a rather large CV (Coefficient ofVariation (i.e., Standard Deviation/Mean)) of 8.5% and isn't centeredabout the set point of 1. FIG. 5b shows the results of applying closedloop feedback on the LTR, the output is centered on the set point of 1and has a CV of 3%.

The invention provides a f feedback control system for independentlycontrolling microfluidic droplet volume and frequency comprising: (a)providing a microfluidic system comprising at least one microfluidicchannel; (b) producing a plurality of fluidic droplets, within a carrierfluid within said microfluidic channel using a drive fluid; (c)assessing the frequency, volume, and flow rate of said plurality ofdroplets using an image sensor; (d) transmitting said assessedfrequencies, volumes, and flow rates of the plurality of droplets fromsaid image sensor to a feedback controller; (e) adjusting a flow rate ofthe carrier fluid using said feedback controller to attain apredetermined droplet frequency set point; and (f) adjusting a flow rateof the drive fluid using said feedback controller to attain apredetermined droplet volume set point; wherein said feedback controlsystem independently determines and controls microfluidic dropletfrequency and volume.

Droplet volume and frequency are intrinsically linked through the law ofmass conservation; droplet frequency multiplied by droplet volume is thedroplet volumetric flow rate. Neglecting any system losses such asleaks, the droplet volumetric flow rate is determined by the drive pumpflow rate. The droplet frequency is a function of many factors such asthe microfluidic nozzle geometry, carrier fluid flow rate, fluidic shearforces, viscosity, and surface tension and will thusly be different fordifferent fluids even when operating under the same pump flow rates.Typically the fluidic system will be initialized with empirically foundpump flow rates starting the system near the desired frequency rate anddroplet volume set point. The first stage of control then starts toadjust the carrier-pump flow rate to move the droplet frequency towardsthe desired set point. Droplet frequency and volume are highlynon-linear as a function of carrier flow rate, but in general,increasing the carrier flow rate will increase the droplet frequency anddecrease droplet volume. Decreasing the carrier flow rate decreases thedroplet frequency and increases the droplet volume. Once the dropletfrequency has settled the second stage of control then adjusts the flowrate to manipulate the droplet volume towards the desired set point.Preferably, the second stage of control adjusts the drive pump flow rateand the resultant drive fluid.

The measurement of absolute droplet volume is of fundamental importance,but traditional methods of measurement require specialized skills in theart and relatively expensive optical instruments. These methods includefluorescence burst analysis and image analysis of projected dropletarea, where the latter requires independent calibration most oftenachieved by the former method. Methods of the invention are easy to use,amenable to automation, and inexpensive to implement. This method can beused in conjunction with the imaging-based control feedback describedabove to create steady streams of droplets of known absolute size andfrequency. The traditional methods are described first, below.

The most accessible measurement related to droplet volume is thevolumetric flow rate,

, of the sample fluid, that is, the liquid phase that forms the dropletsand as opposed to the carrier fluid that surrounds the droplets. Typicalmicrofluidic flow rates between 10 to 10⁴ μL/hr can be measured bynumerous methods including piston displacement and heat transfer. Thus,all that remains to determine the average droplet volume is to measurethe droplet frequency, v, because the average droplet volume, V, equals

$\overset{\_}{V} = {\frac{Q}{v}.}$

This commonly used relationship yields an average droplet size becausethe droplet frequency is determined over an ensemble of droplets.

Droplet frequency poses a more significant measurement challenge.Frequencies often reach ˜10 kHz, requiring a measurement system with avery fast time response. Laser-induced fluorescence is the most commonmethod, taking advantage of the high speed of low light detectors suchas PMTs. In this method, droplets containing fluorophores emit a steadytrain of fluorescence bursts that is readily translated into dropletfrequency by standard Fourier analysis. While quite robust, thisapproach requires familiarity with laser alignment inside a microscopeand it also requires both expensive fluorescence excitation anddetection. Methods of the invention eliminate both of theserequirements.

The invention provides a method called Pulsed Illumination Scanning(PILS). The PILS method is a variant of conventional particle imagevelocimetry (PIV) that has been optimized for steady streams ofregularly spaced droplets. Both approaches measure velocity bymonitoring particle/droplet displacement in between successive imagesseparated by a delay time, ΔT . As an example, in PIV thecross-correlation of two successive images of a field of randomlydispersed particles yields a singular peak corresponding to the uniformdisplacement of all of the particles in the field of view. However,cross-correlation of successive images of regularly spaced dropletsyields a repeating set of peaks because each individual image has a highdegree of autocorrelation. That is, except at the shortest delaysbetween images, it is very difficult to deduce a priori which dropletscorrespond to each other. At very short delays, the percent uncertaintyin the displacement measurement is unacceptably high for mostapplications.

In the PILS method, pairs of successive images are recorded with anincreasing delay in time between images (increasing ΔT). The initial ΔTmust be significantly shorter than the droplet period (the time betweendroplet arrivals, or 1/v) to avoid ambiguity in droplet associationsbetween images. ΔT is then increased gradually to reduce the percenterror in the displacement measurement, but without losing track ofdroplet associations. In fact, ΔT can even significantly exceed thedroplet period so long as the association is maintained. In this manner,the PILS method overcomes the shortcomings of PIV by using both shortdelays to establish associations and long delays to reduce experimentalerror. However, manually stepping between delays can be quite tedious,so a specific PILS method based on Fourier analysis, called ƒPILS isprovided herein, ƒPILS is readily automated.

In the first step of the ƒPILS method, a one-dimensional Fouriertransform of a line-cut along the droplet train (line cut in FIG. 9A)reveals the fundamental droplet frequency (spatial frequency) and itshigher order harmonics (FIG. 9B). The fundamental spatial frequency canbe identified from a single image, but subsequent analysis requires bothimages separated by ΔT to be superimposed. A convenient and inexpensivemethod of superimposing images used here employs short pulses ofillumination from an LED and an extended camera exposure that catchesboth pulses. The amplitude of the fundamental frequency is thenmonitored after each increase in ΔT (FIG. 9C) in the multipleillumination images. The amplitude of the fundamental spatial frequencyoscillates with a period equal to the droplet period. This can beunderstood by considering the case when ΔT equals half the dropletperiod. In this case, the droplets in the second exposure appear halfwayin between the droplets from the first exposure. In effect, thesuperimposed image looks like the droplets have exactly doubled theirfrequency. The new fundamental spatial frequency is now twice theoriginal, and the amplitude of the original frequency is ideally zero.Thus the amplitude of the fundamental spatial frequency oscillatesbetween a maximum at overlap of droplets and a minimum at ½ offsetbetween droplets with a period equal to the droplet period. A secondFourier transform of the dependence of the fundamental frequency on ΔTreveals the desired temporal frequency of the droplets as a singlepronounced peak (FIG. 9D).

The ƒPILS method is low cost, robust, precise, and accurate. The methodonly requires an inexpensive camera that is standard equipment on anydroplet characterization platform, a very inexpensive LED, and a simplepulsed current source to power the LED. The LED pulser used here wasbased on the common and inexpensive PIC microcontroller. The method isalso extremely robust against drift in microscope focus because it isbased on the repetition of features within an image. Even quiteout-of-focus images show excellent repetition. The resolution of themeasurement rivals alternative approaches when many periods of theoscillation in fundamental spatial frequency are observed. In fact, atthe longest ΔT's in FIG. 9C the droplets in the first image havecompletely displaced outside of the field of view in the second image.Such extended ΔT's are impossible with conventional PIV, highlightingthe extra information accessible from a repetitive system. The accuracyof the method is dependent on the uncertainty in ΔT and

. Typically ΔT is very well known, originating from an extremelyaccurate and precise crystal oscillator. Thus, the overall uncertainlyin the measurement is most likely dominated by the error in

.

An example of droplet volume control is shown in FIG. 6. The dropletvolume is detected using either fPILS or inferred from the dropletprojected area as detailed in the droplet detection section. The drivefluid is then automatically adjusted using PID control to control thedroplet volume. It can be seen that droplet diameter and droplet volumeare quite controllable and settle to the desired set point in under 20seconds. FIG. 7 shows the error in the droplet volume is quite low, wellbelow 0.5 pL.

Similarly to droplet volume control, above, the droplet frequency can becontrolled in a straightforward manner. The droplet frequency can bemeasured directly by the ƒPILS method, fluorescence burst analysis, orany other method. Comparison of the measured frequency with the targetset point yields an error signal that can be fed back to a standardcontroller, such as a PID controller. In the preferred control scheme,the carrier flow rate is increased to increase the droplet frequency,and vice versa. Any other method of adjusting the droplet frequency canalso be used.

An example of droplet count control is shown FIG. 8. The current totaldroplet count is detected by integrating the fPILS algorithm output overtime or by integrating the droplet count per image frame over time. Oncethe desired number of droplets has been detected, all plump flow stops,thus stopping droplet formation/release. It can be seen that the dropletrelease/generation count is quite controllable over a large range and ishighly linear.

This present invention provides various methods of droplet detection andanalysis.

“Droplet detection” refers to the identification and selection ofdroplets through an automatic process. In one example, droplet detectionincludes the steps of image acquisition, intensity thresholding, areathresholding, circularity filtering and accumulating the filteredresults. The term “image acquisition” refers to acquiring images ofdroplets. In a preferred aspect of this method, images of droplets areacquired in a microfluidic device. In another preferred aspect of thismethod, images of droplets are acquired in the region of interest (ROI).As used herein, “region of interest” or “ROI” refers to locations in themicrofluidic device where certain behaviors of droplets (e.g., pairing,merging, combination, etc.) occur or where certain actions (e.g.,electrification, mechanical deformation, etc.) are applied to droplets.Preferably, the region of interest or ROI has two ends wherein one endis at the location where droplets enter the ROI and the other end is atthe location where droplets exit the ROI. In a preferred example, theROI is where pairing of droplets occurs. In another preferred example,the ROI is where combination of droplets occurs.

Image acquisition is performed using a device with means to captureimages at a sufficient acquisition rate (e.g., 10 images per second) andexposure time (between 1-10 μs, preferably 5 μs). Alternatively, or inaddition, images are acquired with a digital device (e.g., digitalcamera). The composition of the droplets has a different refractiveindex from that of the surrounding carrier fluid. Thus, due torefraction, the boundary of the droplet has a different brightness,e.g., the boundary of the droplet is darker. Therefore, thecorresponding pixels in the image have different values from those ofthe surrounding pixels.

One method of droplet detection is machine vision, One of ordinary skillin the art of machine vision knows that each image must have sufficientcontrast, focus and resolution to have a robust detection method. Thus,adjusting the optics, illumination and focus to obtain a suitable imageis imperative for droplet detection. The term “accumulated contourdetection” refers to the process in which droplets are detected andcharacterized through multiple image processing filters as follows:

-   -   1. “Intensity threshold” the image at threshold t. In one        example t is initialized to the minimum image intensity value.    -   2. Detect all contours in the threshold image (“contour        detection”).    -   3. Filter contours based on “area thresholding.”    -   4. Filter contours based on “circularity thresholding.”    -   5. Filter contours based on spatial location within the image.    -   6. Accumulate contours into the “contour accumulator image.” For        example, in FIG. 1, an image of droplets in the ROI shows        droplets of two sizes (top panel). The bottom panel shows the        final “contour accumulator image” where grey level intensity        corresponds to the number of times each pixel was added to the        accumulator image. All other pixels were assigned the value “0”,        shown as black.    -   7. Increment the threshold value t.    -   8. Repeat steps 1 though 7 until t reaches the maximum image        intensity value.    -   9. “Intensity threshold” the “contour accumulator image” to        select only droplets with a significant number of votes.

The term “intensity thresholding” refers to the process where the valueof each pixel in an image is compared with a preset value called“threshold value”, and pixels that have a value lower than the thresholdvalue are assigned a designated value, e.g., 0, and pixels that have ahigher value are assigned another designated value, e.g., 1. The outputis called a binary threshold image.

The term “contour detection” refers to the process where the perimeterof connected non-zero pixels is detected. Connectivity defines whichpixels are connected to other pixels. A set of pixels in a binarythreshold image that form a connected group is referred to as an“object” and the perimeter of the “object” is referred to as a“contour”.

The term “area thresholding” refers to the process where the number ofpixels in an area confined by a “contour” is calculated and comparedwith a sot of preset values, and, according to the result from thecomparison, all pixels included in the area are assigned to designatedvalues, e.g., 0 and 1. In one example, areas with the number of pixelseither smaller than a preset value t₁ or bigger than a preset value t₂are assigned a designated value, e.g., 0, to all their pixels, and areaswith the number of pixels no smaller than t₁ and no bigger than t₂ areassigned a designated value, e.g., 1, to all their pixels. Areathresholding detects droplets of sizes within a given range

The term “circularity filtering” refers to the process where formula Iis applied to an area confined by a contour and the resulting value Circis compared with a preset range of values. Only those areas with a Circvalue within the given range are selected. For droplets that arespherical, and, thus, have a circular cross-section, circularityfiltering removes contaminants which often have irregular shapes. informula I, Area and perimeter refer to the total number of pixelsincluded in the area and the total number of pixels present on theboundary of the area, respectively.

$\begin{matrix}{{Circ} = \frac{4{\pi \cdot {Area}}}{{perimeter}^{2}}} & (I)\end{matrix}$

The term “contour accumulation” refers to the process where the resultsof intensity thresholding, contour detection, area thresholding andcircularity filtering are summed into an “accumulator image”. The“accumulator image” keeps count of the number of times each pixel passesthe applied filters. Only pixels that pass the filters many times areactually droplets; this reduces spurious results from illuminationvariations and digital sensor noise.

The term “Droplet classification” refers to the process where dropletsare classified according to their specific properties. Such propertiesinclude, but are not limited to, size (e.g., diameter, perimeter,diagonal, volume, area of cross-section, moments of inertia, etc.),shape (e.g., spherical, elliptical, rectangular, etc.), color,refractive index and extinction coefficient. In one example, thedroplets are classified according to the area of their cross-section.For example, in FIG. 2, cross-sections of droplets are shown, and thedroplets of bigger cross-section area were automatically colored yellow(light shaded) and the droplets of smaller cross-section area werecolored blue (dark shaded).

The invention provides a method to measure every single droplet atnominal generation rates (typically 1 kHz to 10 kHz). Real-timemeasurement of droplet frequency, droplet spacing, effective dropletdiameter, droplet count and nominal bulk fluid flow rates are easilydetected. FIG. 10, shows a trans-illumination scheme but the inventioncan also include any illumination scheme, including but not limited to,epi-illumination and dark-field illumination schemes. The inventionincludes a light source, illumination optics, imaging optics and alinear sensor array. The light source can include ally light source,including but not limited to, LED, laser, incandescent light bulb, fiberoptic bundle, and OLED. Preferably, the light source is an LED. Theillumination optics can include any illumination optics, including butnot limited to, fiber optics, GRIN lens, multiple element lens and lightshaping diffusers. A preferred embodiment is a single plano-convex lenspositioned such that the LED is imaged at infinity. There may be anaperture to control the illuminated field-of-view to illuminate only thearea near the micro-fluidic channel. The imaging optics can include anyimaging optics, including but not limited to, cylindrical lens, fiberoptics, GRIN lens, anamorphic lenses, and multiple element lenses. Apreferred embodiment is a cylindrical lens which images a 2D area fromthe microfluidic device onto a single line of the linear array sensor.There may be an aperture to control optical aberrations and stray light.The linear sensor array can be any linear sensor array. A preferredembodiment is a high speed (>8 MHz) linear array sensor with at least128 pixels and 5 μm pixel size.

In this method, the imaging optics “squeezes” a 2D image into a 1D imageby optically summing what would be the columns of the blue region ofinterest into a single line as shown in FIG. 11. High-speed linear arraysensors with 8 MHz and faster readout rates which provide an inexpensivereplacement for the area-scan CCD imager are readily available. The highreadout rate and linear pixel spacing allows the measurement of dropletparameters with very high precision in time and space respectively.

A single image from the linear sensor array allows the measurement ofdroplet spacing and droplet diameter. As described above and shown inFIG. 11, the output of the linear sensor array clearly shows a periodicsignal representing the droplets as they are positioned within themicrofluidic channel. Droplet position measured in pixels can beconverted to physical position by determining the optical magnificationof the imaging optics. There are many ways to analyze period signals butin one embodiment measurement is as follows: (1) compute the FourierTransform of the 1D image; (2) search for the maximum power signal fromthe transformed data; (3) the droplet spacing corresponds to thetransformed data with maximum power signal. FIG. 12 shows that theaverage droplet spacing is approximately 50 pixels and droplet diameteris approximately 25 pixels.

Taking many samples over time from the linear sensor array allows themeasurement of droplet frequency, droplet count and bulk fluid flowrate. Droplet frequency can be computed by applying the FourierTransform to the time data in a similar manner to the analysis performedon the spatial data described previously. Typical linear sensor arrayshave readout rates at 8 MHz and above, this easily allows themeasurement of each droplet at typical droplet generation rates of up to10 KHz. The droplet count is determined by integrating the dropletfrequency with respect to time. The bulk fluid flow rate is the productof the microfluidic channel cross-sectional area, droplet frequency anddroplet spacing.

The combination of image processing and feedback control provide otherfunctionality beyond droplet control. The inherent stability and knownreference allows for the use of these components to identify anddiagnose issues in the system. The system can be tuned to be robust torun-to-run and intra-run changes in fluid environment, pump controls,and microfluidic device variations. The inherent stability present inthis system that is not present in an uncontrolled system, can be usedto identify system issues or instabilities. Intra- and inter-runpatterns can be used to detect fluidic leaks, restrictions, mechanicalissues, and input conditions.

Another secondary byproduct of image processing is the examination ofdroplets by size across periods of time (i.e., an entire run). Bycreating a histogram of droplets, a new diagnostic tool was madeavailable. Beyond verification of the feedback control operation, thesehistograms can identify issues in pre-emulsified library, fluidicinterference in the chip, and other system issues.

The present invention also provides a Background Subtraction Algorithm.This method provides improvements over the accumulating contourdetection method. Specifically, the Background Subtraction Algorithmimproves CPU utilization as this method is less computer intensive. Itprevents or decreases sensitivity to variations in illumination andfocus quality, when measuring a certain parameter of the detecteddroplet (like size). Moreover, it prevents or decreases noiseamplification when measuring a certain parameter of the detected droplet(like size), due to accumulation of contours of the same droplet.Specifically the Background Subtraction Algorithm is fast, produces lessmeasurement noise and with improved optics, it is possible to use singlethreshold to detect contours.

Specifically, the Background Subtraction Algorithm takes advantages ofthe fact that droplets always appear to be moving, it calculates imagedifference of every two consecutive images. If the absolute value of themaximum difference between images is less then Threshold1, then one ofthe images is selected and saved as a current background image. This isan image of the channel without droplets. Once background image isobtained each new image is also differenced against the currentbackground image and Threshold2 is applied to the difference image.Contours are then obtained from the binary image and filtered using samecircularity, size and position filters described for ContourAccumulation Algorithm. Advantage of the subtraction is that it allowsto remove image of the droplet channel that can make it harder to detectouter contour of the droplet which is less prone to size variations dueto illumination non-uniformity and defocusing.

A microfluidic system of the present invention includes one or moremicrofluidic channels. The terms microfluidic system, microfluidicdevice, microsubstrate, substrate, microchip, and chip are usedinterchangeably herein. The microfluidic system can include at least oneinlet channel, at least one main channel and at least one inlet module.The microfluidic system can further include at least one coalescencemodule, at least one detection module and one or more sorting modules.The sorting module can be in fluid communication with branch channelswhich are in fluid communication with one or more outlet modules(collection module or waste module). For sorting applications, at leastone detection module cooperates with at least one sorting module todivert flow via a detector-originated signal. It shall be appreciatedthat the “modules” and “channels” are in fluid communication with eachother and therefore may overlap; i.e., there may be no clear boundarywhere a module or channel begins or ends. The dimensions of thesubstrate are those of typical microchips, ranging between about 0.5 cmto about 15 cm per side and about 1 micron to about 1 cm in thickness.The microfluidic and specific modules are described in further detail inWO 2006/040551; WO 2006/040554; WO 2004/002627; WO 2004/091763; WO2005/021151; WO 2006/096571; WO 20071089541; WO 2007/081385 and WO2008/063227, each of which is incorporated by reference in its entirety.

The microfluidic substrates of the present invention include channelsthat form the boundary for a fluid. A “channel,” as used herein, means afeature on or in a substrate that at least partially directs the flow ofa fluid. In some cases, the channel may be formed, at least in part, bya single component, e.g., an etched substrate or molded unit. Thechannel can have any cross-sectional shape, for example, circular, oval,triangular, irregular, square or rectangular (having any aspect ratio),or the like, and can be covered or uncovered (i.e., open to the externalenvironment surrounding the channel). In embodiments where the channelis completely covered, at least one portion of the channel can have across-section that is completely enclosed, and/or the entire channel maybe completely enclosed along its entire length with the exception of itsinlet and outlet.

The channels of the invention can be formed, for example by etching asilicon chip using conventional photolithography techniques, or using amicromachining technology called “soft lithography” as described byWhitesides and Xia, Angewandte Chemie International Edition 37, 550(1998).

An open channel generally will include characteristics that facilitatecontrol over fluid transport, e.g., structural characteristics (anelongated indentation) and/or physical or chemical characteristics(hydrophobicity vs. hydrophilicity) and/or other characteristics thatcan exert a force (e.g., a containing force) on a fluid. The fluidwithin the channel may partially or completely fill the channel. In somecases the fluid may be held or confined within the channel or a portionof the channel in some fashion, for example, using surface tension(e.g., such that the fluid is held within the channel within a meniscus,such as a concave or convex meniscus). In an article or substrate, some(or all) of the channels may be of a particular size or less, forexample, having a largest dimension perpendicular to fluid flow of lessthan about 5 mm, less than about 2 mm, less than about 1 mm, less thanabout 500 microns, less than about 200 microns, less than about 100microns, less than about 60 microns, less than about 50 microns, lessthan about 40 microns, less than about 30 microns, less than about 25microns, less than about 10 microns, less than about 3 microns, lessthan about 1 micron, less than about 300 nm, less than about 100 nm,less than about 30 nm, or less than about 10 nm or less in some cases.

A “main channel” is a channel of the device of the invention whichpermits the flow of molecules, cells, small molecules or particles pasta coalescence module for coalescing one or more droplets, and, ifpresent, a detection module for detection (identification) ormeasurement of a droplet and a sorting module for sorting a dropletbased on the detection in the detection module. The main channel istypically in fluid communication with the coalescence, detection and/orsorting modules, as well as, an inlet channel of the inlet module. Themain channel is also typically in fluid communication with an outletmodule and optionally with branch channels, each of which may have acollection module or waste module. These channels permit the flow ofmolecules, cells, small molecules or particles out of the main channel.An “inlet channel” permits the flow of molecules, cells, small moleculesor particles into the main channel. One or more inlet channelscommunicate with one or more means for introducing a sample into thedevice of the present invention. The inlet channel communicates with themain channel at an inlet module.

The microfluidic substrate can also comprise one or more fluid channelsto inject or remove fluid in between droplets in a droplet stream forthe purpose of changing the spacing between droplets.

The channels of the device of the present invention can be of anygeometry as described. However, the channels of the device can comprisea specific geometry such that the contents of the channel aremanipulated, e.g., sorted, mixed, prevent clogging, etc.

A microfluidic substrate can also include a specific geometry designedin such a manner as to prevent the aggregation of biological/chemicalmaterial and keep the biological/chemical material separated from eachother prior to encapsulation in droplets. The geometry of channeldimension can be changed to disturb the aggregates and break them apartby various methods, that can include, but is not limited to, geometricpinching (to force cells through a (or a series of) narrow region(s),whose dimension is smaller or comparable to the dimension of a singlecell) or a barricade (place a series of barricades on the way of themoving cells to disturb the movement and break up the aggregates ofcells).

To prevent material (e.g., cells and other particles or molecules) frontadhering to the sides of the channels, the channels (and coverslip, ifused) may have a coating which minimizes adhesion. The surface of thechannels of the microfluidic device can be coated with any anti-wettingor blocking agent for the dispersed phase. The channel can be coatedwith any protein to prevent adhesion of the biological/chemical sample.Channels can be coated by any means known in the art. For example, thechannels are coated with Teflon®, BSA, PEG-silane and/or fluorosilane inan amount sufficient to prevent attachment and prevent clogging. Inanother example, the channels can be coated with a cyclized transparentoptical polymer obtained by copolymerization of perfluoro (alkenyl vinylethers), such as the type sold by Asahi Glass Co. under the trademarkCytop. In such an example, the coating is applied from a 0.1-0.5 wt %solution of Cytop CTL-809M in CT-Sole 180. This solution can be injectedinto the channels of a microfluidic device via a plastic syringe. Thedevice can then be heated to about 90° C. for 2 hours, followed byheating at 200° C. for an additional 2 hours. In another embodiment, thechannels can be coated with a hydrophobic coating of the type sold byPPG Industries, Inc. under the trademark Aquapel (e.g.,perfluoroalkylalkylsilane surface treatment of plastic and coatedplastic substrate surfaces in conjunction with the use of a silicaprimer layer) and disclosed in U.S. Pat. No. 5,523,162. By fluorinatingthe surfaces of the channels, the continuous phase preferentially wetsthe channels and allows for the stable generation and movement ofdroplets through the device. The low surface tension of the channelwalls thereby minimizes the accumulation of channel cloggingparticulates.

The surface of the channels in the microfluidic device can be alsofluorinated by any means known in the art to prevent undesired wettingbehaviors. For example, a microfluidic device can be placed in apolycarbonate dessicator with an open bottle of(tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane. The dessicatoris evacuated for 5 minutes, and then sealed for 20-40 minutes. Thedessicator is then backfilled with air and removed. This approach uses asimple diffusion mechanism to enable facile infiltration of channels ofthe microfluidic device with the fluorosilane and can be readily scaledup for simultaneous device fluorination.

The fluids described herein are related to the fluids within amicrofluidic device and the fluids used to introduce microdroplets orother items into a microfluidic device.

The microfluidic device of the present invention is capable ofcontrolling the direction and flow of fluids and entities within thedevice. The term “flow” means any movement of liquid or solid through adevice or in a method of the invention, and encompasses withoutlimitation any fluid stream, and any material moving with, within oragainst the stream, whether or not the material is carried by thestream. For example, the movement of molecules, beads, cells or virionsthrough a device or in a method of the invention, e.g. through channelsof a microfluidic chip of the invention, comprises a flow. This is so,according to the invention, whether or not the molecules, beads, cellsor virions are carried by a stream of fluid also comprising a flow, orwhether the molecules, cells or virions are caused to move by some otherdirect or indirect force or motivation, and whether or not the nature ofany motivating force is known or understood. The application of anyforce may he used to provide a flow, including without limitation,pressure, capillary action, electro-osmosis, electrophoresis,dielectrophoresis, optical tweezers, and combinations thereof, withoutregard for any particular theory or mechanism of action, so long asmolecules, cells or virions are directed for detection, measurement orsorting according to the invention. Specific flow forces are describedin further detail herein.

The flow stream in the main channel is typically, but not necessarily,continuous and may be stopped and started, reversed or changed in speed.A liquid that does not contain sample molecules, cells or particles canbe introduced into a sample inlet well or channel and directed throughthe inlet module, e.g., by capillary action, to hydrate and prepare thedevice for use. Likewise, buffer or oil can also be introduced into amain inlet region that communicates directly with the main channel topurge the device (e.g., or “dead” air) and prepare it for use. Ifdesired, the pressure can be adjusted or equalized, for example, byadding buffer or oil to an outlet module.

As used herein, the term “fluid stream” or “fluidic stream” refers tothe flow of a fluid, typically generally in a specific direction. Thefluidic stream may be continuous and/or discontinuous. A “continuous”fluidic stream is a fluidic stream that is produced as a single entity,e. g., if a continuous fluidic stream is produced from a channel, thefluidic stream, after production, appears to be contiguous with thechannel outlet. The continuous fluidic stream is also referred to as acontinuous phase fluid or carrier fluid. The continuous fluidic streammay be laminar or turbulent in some cases. The continuous phase fluidwithin the main channel of the microfluidic device is referred to as thecarrier fluid. The continuous phase fluid outside the main channel ofthe microfluidic device which is used to introduce a sample fluid(either a continuous sample fluid or a discontinuous sample fluid (e.g.,pre-made fluidic droplets) into the microfluidic device is referred toas the drive fluid.

Similarly, a “discontinuous” fluidic stream is a fluidic stream that isnot produced as a single entity. The discontinuous fluidic stream isalso referred to as the dispersed phase fluid or sample fluid. Adiscontinuous fluidic stream may have the appearance of individualdroplets, optionally surrounded by a second fluid. The dispersed phasefluid can include a biological/chemical material. Thebiological/chemical material can be tissues, cells, particles, proteins,antibodies, amino acids, nucleotides, small molecules, andpharmaceuticals. The biological/chemical material can include one ormore labels known in the art. The label can be an optical label, anenzymatic label or a radioactive label. The label can be any detectablelabel, e.g., a protein, a DNA tag, a dye, a quantum dot or a radiofrequency identification tag, or combinations thereof. Preferably thelabel is an optical label. The label can be detected by any means knownin the art. Preferably, the label is detected by fluorescencepolarization, fluorescence intensity, fluorescence lifetime,fluorescence energy transfer, pH, ionic content, temperature orcombinations thereof.

The term “emulsion” refers to a preparation of one liquid distributed insmall globules (also referred to herein as drops, droplets orNanoReactors) in the body of a second liquid. For example, thediscontinuous phase can be an aqueous solution and the continuous phasecan a hydrophobic fluid such as an oil. This is termed a water in oilemulsion. Alternatively, the emulsion may be a oil in water emulsion. Inthat example, the continuous phase can be an aqueous solution and thediscontinuous phase is a hydrophobic fluid, such as an oil (e.g.,decane, tetradecane, or hexadecane). The droplets or globules of oil inan oil in water emulsion are also referred to herein as “micelles”,whereas globules of water in a water in oil emulsion may be referred toas “reverse micelles”.

The fluidic droplets may each be substantially the same shape and/orsize. The droplets may be uniform in size. The shape and/or size can bedetermined, for example, by measuring the average diameter or othercharacteristic dimension of the droplets. The “average diameter” of aplurality or series of droplets is the arithmetic average of the averagediameters of each of the droplets. Those of ordinary skill in the artwill be able to determine the average diameter (or other characteristicdimension) of a plurality or series of droplets, for example, usinglaser light scattering, microscopic examination, or other knowntechniques. The diameter of a droplet, in a non-spherical droplet, isthe mathematically-defined average diameter of the droplet, integratedacross the entire surface. The average diameter of a droplet (and/or ofa plurality or series of droplets) may be, for example, less than about1 mm, less than about 500 micrometers, less than about 200 micrometers,less than about 100 micrometers, less than about 75 micrometers, lessthan about 50 micrometers, less than about 25 micrometers, less thanabout 10 micrometers, or less than about 5 micrometers in some cases.The average diameter may also be at least about 1 micrometer, at leastabout 2 micrometers, at least about 3 micrometers, at least about 5micrometers, at least about 10 micrometers, at least about 15micrometers, or at least about 20 micrometers in certain cases.

As used herein, the term “NanoReactor” and its plural encompass theterms “droplet”, “nanodrop”, “nanodroplet”, “microdrop” or“microdroplet” as defined herein, as well as an integrated system forthe manipulation and probing of droplets, as described in detail herein.Nanoreactors as described herein can be 0.1-1000 μm (e.g., 0.1, 0.2 . .. 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 . . . 1000), or any size withinthis range. Droplets at these dimensions tend to conform to the size andshape of the channels, while maintaining their respective volumes. Thus,as droplets move from a wider channel to a narrower channel they becomelonger and thinner, and vice versa.

The microfluidic substrate of this invention most preferably generateround, highly uniform, monodisperse droplets (<1.5% polydispersity).Droplets and methods of forming monodisperse droplets in microfluidicchannels is described in WO 2006/040551; WO 2006/040554; WO 2004/002627;WO 2004/091763; WO 2005/021151; WO 2006/096571; WO 2007/089541; WO2007/081385 and WO 2008/063227.

The droplet forming liquid is typically an aqueous buffer solution, suchultrapure water (e.g., 18 mega-ohm resistivity, obtained, for example bycolumn chromatography), 10 mM Tris HCl and 1 mM EDTA (TE) buffer,phosphate buffer saline (PBS) or acetate buffer. Any liquid or bufferthat is physiologically compatible with the population of molecules,cells or particles to be analyzed and/or sorted can be used. The fluidpassing through the main channel and in which the droplets are formed isone that is immiscible with the droplet forming fluid. The fluid passingthrough the main channel can be a non-polar solvent, decane (e.g.,tetradecane or hexadecane), fluorocarbon oil, silicone oil or anotheroil (for example, mineral oil).

The droplet may also contain biological/chemical material (e.g.,molecules, cells, or other particles) for combination, analysis and/orsorting in the device. The droplets of the dispersed phase fluid cancontain more than one particle or can contain no more than one particle.

Droplets of a sample fluid can be formed within the inlet module on themicrofluidic device or droplets (or droplet libraries) can be formedbefore the sample fluid is introduced to the microfluidic device (“offchip” droplet formation). To permit effective interdigitation,coalescence and detection, the droplets comprising each sample to beanalyzed must be monodisperse. As described in more detail herein, inmany applications, different samples to be analyzed are contained withindroplets of different sizes. Droplet size must be highly controlled toensure that droplets containing the correct contents for analysis andcoalesced properly. As such, the present invention provides devices andmethods for forming droplets and droplet libraries.

The fluids used in the invention may contain one or more additives, suchas agents which reduce surface tensions (surfactants). Surfactants caninclude Tween, Span, fluorosurfactants, and other agents that aresoluble in oil relative to water. In some applications, performance isimproved by adding a second surfactant to the aqueous phase. Surfactantscan aid in controlling or optimizing droplet size, flow and uniformity,for example by reducing the shear force needed to extrude or injectdroplets into an intersecting channel. This can affect droplet volumeand periodicity, or the rate or frequency at which droplets break offinto an intersecting channel. Furthermore, the surfactant can serve tostabilize aqueous emulsions in fluorinated oils from coalescing. Thepresent invention provides compositions and methods to stabilize aqueousdroplets in a fluorinated oil and minimize the transport of positivelycharged reagents (particularly, fluorescent dyes) from the aqueous phaseto the oil phase.

The droplets may be coated with a surfactant. Preferred surfactants thatmay be added to the continuous phase fluid include, but are not limitedto, surfactants such as sorbitan-based carboxylic acid esters (e.g., the“Span” surfactants, Fluka Chemika), including sorbitan monolaurate (Span20), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60)and sorbitan monooleate (Span 80), and perfluorinated polyethers (e.g.,DuPont Krytox 157 FSL, FSM, and/or FSH). Other non-limiting examples ofnon-ionic surfactants which may be used include polyoxyethylenatedalkylphenols (for example, nonyl-, p-dodecyl-, and dinonylphenols),polyoxyethylenated straight chain alcohols, polyoxyethylenatedpolyoxypropylene glycols, polyoxyethylenated mercaptans, long chaincarboxylic acid esters (for example, glyceryl and polyglycerl esters ofnatural fatty acids, propylene glycol, sorbitol, polyoxyethylenatedsorbitol esters, polyoxyethylene glycol esters, etc.) and alkanolamines(e.g., diethanolamine-fatty acid condensates and isopropanolamine-fattyacid condensates). In addition, ionic surfactants such as sodium dodecylsulfate (SDS) may also be used. However, such surfactants are generallyless preferably for many embodiments of the invention. For instance, inthose embodiments where aqueous droplets are used as nanoreactors forchemical reactions (including biochemical reactions) or are used toanalyze and/or sort biomaterials, a water soluble surfactant such as SDSmay denature or inactivate the contents of the droplet.

The continuous phase fluid (carrier fluid and the drive fluid) can be anoil (e.g., decant, tetradecane or hexadecane) or fluorocarbon oil thatcontains a surfactant (e.g., a non-ionic surfactant such as a Spansurfactant) as an additive (preferably between about 0.2 and 5% byvolume, more preferably about 2%). A user can preferably cause thecarrier fluid to flow through channels of the microfluidic device sothat the surfactant in the carrier fluid coats the channel walls.

Fluorocarbon oil continuous phases are well-suited as the continuousphase for aqueous droplet libraries for a number of reasons. Fluorousoils are both hydrophobic and lipophobic. Therefore, they have lowsolubility for components of the aqueous phase and they limit moleculardiffusion between droplets. Also, fluorous oils present an inertinterface for chemistry and biology within droplets. In contrast tohydrocarbon or silicone oils, fluorous oils do not swell PDMS materials,which is a convenient material for constructing microfluidic channels.Finally, fluorocarbon oils have good solubility for gases, which isnecessary for the viability of encapsulated cells.

Combinations of surfactant(s) and oils must be developed to facilitategeneration, storage, and manipulation of droplets to maintain the uniquechemical/biochemical/biological environment within each droplet of adiverse library. Therefore, the surfactant and oil combination must (1)stabilize droplets against uncontrolled coalescence during the dropforming process and subsequent collection and storage, (2) minimizetransport of any droplet contents to the oil phase and/or betweendroplets, and (3) maintain chemical and biological inertness withcontents of each droplet (e.g., no adsorption or reaction ofencapsulated contents at the oil-water interface, and no adverse effectson biological or chemical constituents in the droplets). In addition tothe requirements on the droplet library function and stability, thesurfactant-in-oil solution must be coupled with the fluid physics andmaterials associated with the platform. Specifically, the oil solutionmust not swell, dissolve, or degrade the materials used to construct themicrofluidic chip, and the physical properties of the oil (e.g.,viscosity, boiling point, etc.) must be suited for the flow andoperating conditions of the platform.

Droplets formed in oil without surfactant are not stable to permitcoalescence, so surfactants must be dissolved in the fluorous oil thatis used as the continuous phase for the emulsion library. Surfactantmolecules are amphiphilic—part of the molecule is oil soluble, and partof the molecule is water soluble. When a water-oil interface is formedat the nozzle of a microfluidic chip for example in the inlet moduledescribed herein, surfactant molecules that are dissolved in the oilphase adsorb to the interface. The hydrophilic portion of the moleculeresides inside the droplet and the fluorophilic portion of the moleculedecorates the exterior of the droplet. The surface tension of a dropletis reduced when the interface is populated with surfactant, so thestability of an emulsion is improved. In addition to stabilizing thedroplets against coalescence, the surfactant should be inert to thecontents of each droplet and the surfactant should not promote transportof encapsulated components to the oil or other droplets.

The invention can use pressure drive flow control, e.g., utilizingvalves and pumps, to manipulate the flow of cells, particles, molecules,enzymes or reagents in one or more directions and/or into one or morechannels of a microfluidic device. However, other methods may also beused, alone or in combination with pumps and valves, such aselectro-osmotic flow control, electrophoresis and dielectrophoresis asdescribed in Fulwyer, Science 156, 910 (1974); Li and Harrison,Analytical Chemistry 69, 1564 (1997); Fiedler, et al. AnalyticalChemistry 70, 1909-1915 (1998) and U.S. Pat. No. 5,656,155. Applicationof these techniques according to the invention provides more rapid andaccurate devices and methods for analysis or sorting, for example,because the sorting occurs at or in a sorting module that can be placedat or immediately after a detection module. This provides a shorterdistance for molecules or cells to travel, they can move more rapidlyand with less turbulence, and can more readily be moved, examined, andsorted in single file, i.e., one at a time.

Positive displacement pressure driven flow is a preferred way ofcontrolling fluid flow and dielectrophoresis is a preferred way ofmanipulating droplets within that flow. The pressure at the inlet modulecan also be regulated by adjusting the pressure on the main and sampleinlet channels, for example, with pressurized syringes feeding intothose inlet channels. By controlling the pressure difference between theoil and water sources at the inlet module, the size and periodicity ofthe droplets generated may be regulated. Alternatively, a valve may beplaced at or coincident to either the inlet module or the sample inletchannel connected thereto to control the flow of solution into the inletmodule, thereby controlling the size and periodicity of the droplets.Periodicity and droplet volume may also depend on channel diameter, theviscosity of the fluids, and shear pressure. Examples of drivingpressures and methods of modulating flow are as described in WO2006/040551; WO 2006/040554; WO 2004/002627; WO 2004/091763; WO2005/021151; WO 2006/096571; WO 2007/089541; WO 2007/081385 and WO2008/063227; U.S. Pat. No. 6,540,895 and U.S. Patent ApplicationPublication Nos. 20010029983 and 20050226742

The microfluidic device of the present invention may include one or moreinlet modules. An “inlet module” is an area of a microfluidic substratedevice that receives molecules, cells, small molecules or particles foradditional coalescence, detection and/or sorting. The inlet module cancontain one or more inlet channels, wells or reservoirs, openings, andother features which facilitate the entry of molecules, cells, smallmolecules or particles into the substrate. A substrate may contain morethan one inlet module if desired. Different sample inlet channels cancommunicate with the main channel at different inlet modules.Alternately, different sample inlet channels can communication with themain channel at the same inlet module. The inlet module is in fluidcommunication with the main channel. The inlet module generallycomprises a junction between the sample inlet channel and the mainchannel such that a solution of a sample (i.e., a fluid containing asample such as molecules, cells, small molecules (organic or inorganic)or particles) is introduced to the main channel and forms a plurality ofdroplets. The sample solution can be pressurized. The sample inletchannel can intersect the main channel such that the sample solution isintroduced into the main channel at an angle perpendicular to a streamof fluid passing through the main channel. For example, the sample inletchannel and main channel intercept at a T-shaped junction; such that thesample inlet channel is perpendicular (90 degrees) to the main channel.However, the sample inlet channel can intercept the main channel at anyangle, and need not introduce the sample fluid to the main channel at anangle that is perpendicular to that flow. The angle between intersectingchannels is in the range of from about 60 to about 120 degrees,Particular exemplary angles are 45, 60, 90, and 120 degrees.

Embodiments of the invention are also provided in which there are two ormore inlet modules producing droplets of samples into the main channel.For example, a first inlet module may produce droplets of a first sampleinto a flow of fluid in the main channel and a second inlet module mayproduce droplets of a second sample into the flow of fluid in mainchannel, and so forth, The second inlet module is preferably downstreamfrom the first inlet module (e.g., about 30 μm). The fluids producedinto the two or more different inlet modules can comprise the same fluidor the same type of fluid (e.g., different aqueous solutions). Forexample, droplets of an aqueous solution containing an enzyme areproduced into the main channel at the first inlet module and droplets ofaqueous solution containing a substrate for the enzyme are produced intothe main channel at the second inlet module. Alternatively, the dropletsproduced at the different inlet modules may be droplets of differentfluids which may be compatible or incompatible. For example, thedifferent droplets may be different aqueous solutions, or producedintroduced at a first inlet module may be droplets of one fluid (e.g.,an aqueous solution) whereas droplets produced at a second inlet modulemay be another fluid (e.g., alcohol or oil). The terms “produced” or“producing” are meant to describe forming, generating, or creatingdroplets from a continuous sample source. Moreover, the term producingencompasses introducing pre-formed droplets (e.g., droplets made offchip) into a microfluidic channel in a microfluidic device.

1-20. (canceled)
 21. A system for controlling droplet flow rate, thesystem comprising: a microfluidic device comprising a microfluidicchannel and a merge zone, the microfluidic channel comprising a firststream of droplets and a second stream of droplets; a sensor configuredto detect flow rates of the first and second streams of droplets in themicrofluidic channel; and a controller operably connected to the sensor,the controller configured to adjust the flow rates relative to eachother to cause the first stream of droplets to intercalate with thesecond stream of droplet in the merge zone.
 22. The system of claim 21,wherein the controller is configured to adjust the flow rates byadjusting a pressure of one or more fluids.
 23. The system of claim 21,wherein the first and second streams of droplets comprise dropletsdiffering in size, color, refractive index, or extinction coefficient.24. The system of claim 21, wherein the first and second streams ofdroplets contain different biological, biochemical, or chemicalentities.
 25. The system of claim 21, wherein a frequency ratio of thefirst stream of droplets to the second stream of droplets is selectedfrom the group consisting of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, and 1:10.
 26. The system of claim 25, wherein the frequency ratioof the first stream of droplets to the second stream of droplets is 1:1.27. The system of claim 21, wherein droplets of the first stream have amaximum cross-sectional dimension of less than about 100 microns. 28.The system of claim 21, wherein droplets of the second stream have amaximum cross-sectional dimension of less than about 100 microns. 29.The system of claim 21, wherein the controller adjusts the flow rates ofone or both streams of droplets to maintain an equal flow rate.
 30. Thesystem of claim 21, further comprising a drive infusion systemconfigured to drive the streams of droplets through the microfluidicchannel, the drive infusion system selected from the group consisting ofa pump, a pressure head, and an electro-kinetic drive.
 31. The system ofclaim 30, wherein drive infusion system is in communication with thecontroller.